骨形成蛋白2活性多肽体外定向诱导骨髓间充质干细胞向成骨方向分化的剂量依赖性研究  

DOSE-DEPENDENCE OF BONE MORPHOGENETIC PROTEIN 2-DERIVED PEPTIDE ON OSTEOGENIC INDUCTION IN MARROW MESENCHYMAL STEM CELLS IN VITRO

作　　者：段智霞[1] 郑启新[1] 郭晓东[1] 袁泉[1] 陈顺广[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科

出　　处：《中国修复重建外科杂志》2007年第10期1118-1122,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30170270;30200063;30470483);武汉市青年科技晨光计划资助项目(20045006071-10)~~

摘　　要：目的探讨自行合成的骨形成蛋白2(bone morphogenetic protein2,BMP-2)活性多肽对大鼠骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)体外定向诱导成骨的能力,评价BMP-2活性多肽的成骨诱导性及诱导成骨的剂量依赖性。方法取4周龄Wistar大鼠分离培养MSCs,传至第3代时改用条件培养基,培养基中分别加入浓度为300、200、100、50及0μg/ml的BMP-2活性多肽,并依次记为A、B、C、D和E组。细胞培养至5、10、15及20d,检测碱性磷酸酶(alkaline phosphatase,ALP)活性,测定钙含量,采用实时荧光定量PCR(fluorescent quantitativePCR,FQ-PCR)检测型胶原、骨桥蛋白(osteopontin,OPN)及骨钙素(osteocalcin,OCN)mRNA的表达,检测不同浓度BMP-2活性多肽体外诱导成骨的能力。结果倒置相差显微镜下观察,用条件培养基培养3～4d后,培养细胞由长梭形转变为短梭形或多角形。随条件培养基中BMP-2活性多肽浓度的增加,细胞发生成骨样改变的时间提前。ALP活性和钙含量检测显示,A组和B组较其他组增加明显,且差异有统计学意义(P<0.05),但A、B组间比较差异无统计学意义(P>0.05)。FQ-PCR检测发现不同浓度条件培养基培养14d后,型胶原、OPN和OCN mRNA在各组均有表达。Ct值表明其表达量的大小顺序为A、B、C、D组,E组无表达,A组和B组的Ct值明显大于C组和D组,差异有统计学意义(P<0.05),但A组和B组之间差异无统计学意义(P>0.05)。结论BMP-2活性多肽能有效地促进MSCs向成骨方向分化,其诱导作用存在剂量依赖关系,最佳诱导剂量为200μg/ml。Objective To investigate the effect of the synthetic bone morphogenetic protein 2 （BMP-2）-derived peptide on the osteogenic induction in the marrow mesenchymal stem cells （MSCs）and to evaluate the osteoinductivity and dose-dependence of the BMP-2-derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added： 300,200, 100, 50, and 0 μg/ml, respectively （Groups A-E）. The activity of alkaline phosphatase （ALP）and the amount of calcium deposition were meassured at 5,10,15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） was performed to measure the mRNA expressions of collagen type Ⅰ , osteopontin （OPN）, and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations. Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（P〈0. 05）. However, there was no significant difference between Group A and Group B （P〉0. 05）. The result of FQ-PCR showed that after MSCs were cultured in the different doses of the conditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ ,OPN and OCN were at higher levels. An increasing order in the level of the cycle threshold （Ct） was found in the following groups： A）B）C）D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B than in Groups C and D（P〈0. 05）. However, there

关 键 词：骨形成蛋白2活性多肽 骨髓间充质干细胞 成骨诱导 剂量依赖性 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]